Differential regulation of the pituitary gonadotropin (FSH and LH) gene expression by GnRH and other regulators of the hypothalamic-pituitary-gonadal axis is key in the control of reproductive functions. GnRH is released by the hypothalamus in a pulsatile manner. Dysregulation of gonadotropin expression due to alterations in GnRH pulse release causes reproductive disorders such as polycystic ovary syndrome and endometriosis, as well as hormone-dependent cancers. Our recent identification of several putative autocrine regulators of FSH, including INHA, VGF/NERP-1, and GDF9, supports the contribution of an exosignaling network to gonadotropin gene regulation. Furthermore, by developing simple mathematical models and a high throughput GnRH pulse experimental system, we have demonstrated that FSHb responds to changes in both average GnRH concentration and GnRH pulse frequency in the gonadotrope L?T2 cell line. Unraveling the molecular mechanisms of gonadotropin gene regulation requires not only studies in primary gonadotropes and in vivo models, but also dissection of the cellular heterogeneity that is concealed in the population average. Previous reports and our preliminary single-cell (SC) transcriptome data obtained from mouse pituitary cells strongly suggest that gonadotropes are functionally heterogeneous cells. We propose the following Specific Aims: 1. Elucidate the gonadotrope gene regulatory network from single cell transcriptome maps of mouse pituitary. Using the SC stabilization technology developed in our laboratory, pituitaries from both male and female mice will be subjected to Gel in EMulsion droplet-based SC RNA-sequencing (GEM drop-seq). This will allow the characterization of pituitary and gonadotrope subtypes. Customized computational methods will be employed to examine gene expression changes occurring during estrous cycle, identify regulatory modules and factor enrichment, and infer the gene regulatory networks underlying gonadotropin gene regulation. Hypotheses emerging from this investigation will be tested in vivo by analyzing SC transcriptomes of pituitaries from hormone-treated or KO animals. 2. Elucidate gene regulatory circuits and mechanisms underlying Fshb regulation by extracellular factors. Gene responses of L?T2 cells to GnRH pulse stimulation in the presence or absence of candidate modulators will be initially assessed by SC droplet qPCR, followed by a SC Drop-seq analysis of selected samples. Computational analysis of the data and their comparison to mouse pituitary data obtained in Aim 1 will allow the development of models of Fshb regulation. These models will be refined experimentally by combining targeted siRNA knockdown with pulse response studies, mapping TF binding i.e. chromatin accessibility genome-wide by ATAC-seq, and generating new hypotheses that will be testable using in vivo models in Aim 1. Overall, the proposed studies will generate a valuable data resource of pituitary SC transcriptomes, provide important insight into the molecular heterogeneity of gonadotropes, and contribute to a deeper and thorough understanding of the mechanisms of Fshb regulation. !